Eml4-alk translocations in lung cancer

ABSTRACT

The present disclosure relates to methods for the diagnosis and evaluation of neoplastic disorders, particularly non-small cell lung cancer. Assays are described in which patient test samples are analyzed for the presence of one or more specific EML4-ALK fusion genes associated with neoplastic disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Applications61/289,234, filed Dec. 22, 2009 and 61/301,551, filed Feb. 4, 2010.

FIELD OF THE INVENTION

The present invention relates generally to the field of medicaldiagnostics. In particular, the present technology relates to methods ofdetecting genetic mutations associated with cancer.

BACKGROUND OF THE INVENTION

The following description is provided to assist the understanding of thereader. None of the information provided or references cited is admittedto be prior art to the present invention.

Many cancers are characterized by disruptions in cellular signalingpathways that lead to aberrant control of cellular processes, or touncontrolled growth and proliferation of cells. These disruptions areoften caused by changes in the activity of particular signalingproteins, such as kinases. Among these cancers are solid tumors, likenon-small cell lung cancer (NSCLC). NSCLC is the leading cause of cancerdeath in the United States, and accounts for about 87% of all lungcancers. There are about 151,000 new cases of NSCLC in the United Statesannually, and it is estimated that over 120,000 patients will dieannually from the disease in the United States alone. NSCLC, whichcomprises three distinct subtypes, is often only detected after it hasmetastasized, and thus the mortality rate is 75% within two years ofdiagnosis.

It is known that gene deletions and/or translocations resulting inkinase fusion proteins with aberrant signaling activity can directlylead to certain cancers. For example, it has been demonstrated that theBCR-ABL oncoprotein, a tyrosine kinase fusion protein, is a causativeagent in human chronic myelogenous leukemia (CML). The BCR-ABLoncoprotein, which is found in at least 90-95% of CML cases, isgenerated by the translocation of gene sequences from the c-ABL proteintyrosine kinase on chromosome 9 into BCR sequences on chromosome 22,producing the so-called Philadelphia chromosome. See, e.g. Kurzock etal., N Engl. J. Med. 319: 990-998 (1988). The translocation is alsoobserved in acute lymphocytic leukemia and NSCLC cases.

EML4-ALK is a fusion-type protein tyrosine kinase that is generated inhuman non-small-cell lung cancer (NSCLC) and other cancers as a resultof a recurrent chromosome inversion, inv (2)(p21p23). EML4 (echinodermmicrotubule-associated protein like protein 4) is a cytoplasmic proteinwith a molecular weight of 120,000, which is highly expressed in the Mphase of the cell cycle. The human EML4 gene encodes a polypeptide with981 amino acids and has 23 exons. The EML4 protein has a basic region atthe amino terminus, as with other members of the EML family, and furtherhas carboxyl-terminal WD domains. The function of EML4 in cells is notknown. However, according to a recent report, EML4 may participate inmicrotubule formation.

ALK (Anaplastic Lymphoma Kinaseis receptor tyrosine kinase) is a proteinthat has a transmembrane domain in the central part, a carboxyl-terminaltyrosine kinase region, and an amino-terminal extracellular domain. TheALK gene has 30 exons and encodes a polypeptide with 1,620 amino acids.The ALK gene has been reported to participate in the development orfunctioning of the nervous system. Full-length ALK expression has beenreported in some cancer cells of ectodermal origin, such asneuroblastoma, glioblastoma, breast cancer, and melanoma.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for diagnosing aneoplastic disorder or susceptibility to a neoplastic disordercomprising detecting the presence of a EML4-ALK gene fusion in a samplefrom a subject, wherein the EML4-ALK gene fusion is a fusion betweenexon 17 or intron 17 of EML4 and intron 19 of ALK, and diagnosing thesubject as having or being susceptible to a neoplastic disorder when thegene fusion is present.

In another aspect, the invention provides a method of determining theEML4-ALK gene fusion status of a human by: (a) determining the presenceor absence of the E17;ins30A20 or E17ins30;ins65A20 gene fusion in bothalleles of the of the EML4-ALK fusion gene of the human in a nucleicacid sample obtained from the human, and (b) identifying the human (i)as being homozygous for E17;ins30A20 or E17ins30;ins65A20 gene fusion inthe EML4-ALK fusion gene when one of the gene fusions is present in bothalleles of the EML4-ALK fusion gene, or (ii) as being heterozygous forthe E17;ins30A20 or E17ins30;ins65A20 gene fusion in the EML4-ALK fusiongene when one of the gene fusions is present in one of the alleles ofthe EML4-ALK fusion gene, or (iii) as having no alteration in theEML4-ALK fusion gene caused by the E17;ins30A20 or E17ins30;ins65A20gene fusion when each of the gene fusions is absent from both alleles ofthe EML4-ALK fusion gene.

In some embodiments, the neoplastic disorder is non small cell lungcancer.

In some embodiments, the EML4-ALK gene fusion is E17;ins30A20 orE17ins30;ins65A20. An EML4-ALK gene fusion may be detected by amplifyingSEQ ID NO: 1, SEQ ID NO: 2, or a diagnostic fragment thereof. In oneembodiment, the method includes detecting one or more additionalEML4-ALK gene fusions in a sample from the subject. For instance, theone or more additional EML4-ALK gene fusions may be selected from thegroup consisting of: variant 1, variant 2, variant 3a, variant 3b,variant 4, variant 5a, variant 5b, variant 6, and variant 7.

Suitable samples for assessment of EML4-ALK fusions include, forexample, plasma, serum, and biopsy tissue (e.g., a lung biopsy sample).

The EML4-ALK fusion may be detected by assessing sample nucleic acid byPCR, RT-PCR, and/or nucleic acid hybridization. In one embodiment, thesample is amplified by reverse transcriptase polymerase chain reaction(RT-PCR). In one embodiment, the amplifying employs a detectably labeledprimer. In one embodiment, the detecting is accomplished withelectrophoresis. In one embodiment, the detecting is accomplished usinga real-time PCR-based detection system, such as TaqMan®.

The present invention also provides a method for diagnosing a neoplasticdisorder or susceptibility to a neoplastic disorder by detecting thepresence or absence of an EML4-ALK fusion protein in a sample from asubject, wherein the EML4-ALK fusion protein is a fusion between exon 17or intron 17 of EML4 and intron 19 of ALK, and diagnosing the subjecthas having or being susceptible to a neoplastic disorder when the fusionprotein is present. In one embodiment, the fusion protein contains thesequence of SEQ ID NO: 29 or 30.

The invention also provides kits for detecting a EML4-ALK fusionmutations which include one or more oligonucleotides (e.g., a primer)for amplifying a fragment of a nucleic acid sample which contains theE17;ins30A20 or E17ins30;ins65A20 mutation, if present. In oneembodiment, at least one of the primers contains the sequence of SEQ IDNOs: 19 and 25, or complements thereof. Optionally, the kit furthercontains one or more mutation-specific oligonucleotide probes. In arelated aspect, the invention provides a kit containing at least oneoligonucleotide having the sequence of SEQ ID NOs: 19 or 25-28, orcomplements thereof.

In another aspect, the invention provides an isolated polynucleotidesubstantially identical to SEQ ID NO: 1 or SEQ ID NO: 2 or complementsthereof. Other useful a substantially purified polynucleotides includethose having the sequence of any of SEQ ID NOs: 3-25 or complementsthereof.

The definitions of certain terms as used in this specification areprovided below. Unless defined otherwise, all technical and scientificterms used herein generally have the same meaning as commonly understoodby one of ordinary skill in the art to which this invention belongs.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a nucleic acid”includes a combination of two or more nucleic acids, and the like.

The term “amplification” or “amplify” as used herein means one or moremethods known in the art for copying a target nucleic acid, therebyincreasing the number of copies of a selected nucleic acid sequence.Amplification may be exponential or linear. A target nucleic acid may beeither DNA or RNA. The sequences amplified in this manner form an“amplicon.” While the exemplary methods described hereinafter relate toamplification using the polymerase chain reaction (“PCR”), numerousother methods are known in the art for amplification of nucleic acids(e.g., isothermal methods, rolling circle methods, etc.). The skilledartisan will understand that these other methods may be used either inplace of, or together with, PCR methods. See, e.g., Saiki,“Amplification of Genomic DNA” in PCR Protocols, Innis et al., Eds.,Academic Press, San Diego, Calif. 1990, pp. 13-20; Wharam et al.,Nucleic Acids Res., 2001, 29(11):E54-E54; Hafner et al., Biotechniques2001, 30(4):852-6, 858, 860; Zhong et al., Biotechniques, 2001,30(4):852-6, 858, 860.

The term “complement” as used herein means the complementary sequence toa nucleic acid according to standard Watson/Crick base pairing rules. Acomplement sequence can also be a sequence of RNA complementary to theDNA sequence or its complement sequence, and can also be a cDNA. Theterm “substantially complementary” as used herein means that twosequences hybridize under stringent hybridization conditions. Theskilled artisan will understand that substantially complementarysequences need not hybridize along their entire length. In particular,substantially complementary sequences comprise a contiguous sequence ofbases that do not hybridize to a target or marker sequence, positioned3′ or 5′ to a contiguous sequence of bases that hybridize understringent hybridization conditions to a target or marker sequence.

As used herein the terms “diagnose” or “diagnosis” or “diagnosing” referto distinguishing or identifying a disease, syndrome or condition oridentifying a person having a particular disease, syndrome or condition.In illustrative embodiments of the invention, assays are used todiagnose a neoplastic disorder, such as NSCLC, in a subject based on ananalysis of a sample.

As used herein, the term “EML4-ALK gene fusion” refers to an aberrantgene rearrangement between the EML4 gene and the ALK gene on chromosome2. The term EML4-ALK fusion also refers to the polypeptide formed froman inversion on human chromosome 2 in which the EML4 gene is fused tothe ALK gene and that sequence is translated to form an aberrantprotein. In exemplary embodiments, the EML4-ALK gene fusion is theEML4-ALK protein variant 8a or 8b, or encoding nucleic acids.

As used herein, the term “hybridize” or “specifically hybridize” refersto a process where two complementary nucleic acid strands anneal to eachother under appropriately stringent conditions. Hybridizations aretypically conducted with probe-length nucleic acid molecules. Nucleicacid hybridization techniques are well known in the art. Those skilledin the art understand how to estimate and adjust the stringency ofhybridization conditions such that sequences having at least a desiredlevel of complementarity will stably hybridize, while those having lowercomplementarity will not. For examples of hybridization conditions andparameters, see, e.g., Sambrook, et al., 1989, Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview,N.Y.; Ausubel, F. M. et al. 1994, Current Protocols in MolecularBiology. John Wiley & Sons, Secaucus, N.J.

By “isolated”, when referring to a nucleic acid (e.g., anoligonucleotide such as RNA, DNA, or a mixed polymer) is meant a nucleicacid that is apart from a substantial portion of the genome in which itnaturally occurs and/or is substantially separated from other cellularcomponents which naturally accompany such nucleic acid. For example, anynucleic acid that has been produced synthetically (e.g., by serial basecondensation) is considered to be isolated. Likewise, nucleic acids thatare recombinantly expressed, cloned, produced by a primer extensionreaction (e.g., PCR), or otherwise excised from a genome are alsoconsidered to be isolated.

As used herein, a “fragment” means a linear segment of a targetpolynucleotide that is at least about 15, 20, 25, 30, 35, 40, 45, 50,75, 100, 200, 300, 400, 500, 1000 contiguous nucleotides or more inlength.

As used herein, the term “neoplastic disorder” refers to cancers of anykind and origin and precursor stages thereof. Accordingly, the term“neoplastic disorder” includes the subject matter identified by theterms “neoplasia”, “neoplasm”, “cancer”, “precancer” or “tumor”.Neoplastic disorders to which the methods of the present invention maybe applied include but are not limited to, neoplastic lesions of therespiratory tract, such as non-small cell lung cancer.

As used herein, “nucleic acid” refers broadly to segments of achromosome, segments or portions of DNA, cDNA, and/or RNA. Nucleic acidmay be derived or obtained from an originally isolated nucleic acidsample from any source (e.g., isolated from, purified from, amplifiedfrom, cloned from, or reverse transcribed from sample DNA or RNA).

As used herein, the term “oligonucleotide” refers to a short polymercomposed of deoxyribonucleotides, ribonucleotides or any combinationthereof. Oligonucleotides are generally between about 10 and about 100nucleotides in length. Oligonucleotides are typically 15 to 70nucleotides long, with 20 to 26 nucleotides being the most common. Anoligonucleotide may be used as a primer or as a probe.

An oligonucleotide is “specific” for a nucleic acid if theoligonucleotide has at least 50% sequence identity with a portion of thenucleic acid when the oligonucleotide and the nucleic acid are aligned.An oligonucleotide that is specific for a nucleic acid is one that,under the appropriate hybridization or washing conditions, is capable ofhybridizing to the target of interest and not substantially hybridizingto nucleic acids which are not of interest. Higher levels of sequenceidentity are preferred and include at least 75%, at least 80%, at least85%, at least 90%, or at least 95% sequence identity.

As used herein, a “primer” for amplification is an oligonucleotide thatspecifically anneals to a target or marker nucleotide sequence. The 3′nucleotide of the primer should be identical to the target or markersequence at a corresponding nucleotide position for optimal primerextension by a polymerase. As used herein, a “forward primer” is aprimer that anneals to the anti-sense strand of double stranded DNA(dsDNA). A “reverse primer” anneals to the sense-strand of dsDNA.

As used herein, the term “sample” or “test sample” refers to any liquidor solid material containing nucleic acids. In suitable embodiments, atest sample is obtained from a biological source (i.e., a “biologicalsample”), such as cells in culture or a tissue sample from an animal,most preferably, a human. In an exemplary embodiment, the sample is abiopsy sample.

“Target nucleic acid” as used herein refers to segments of a chromosome,a complete gene with or without intergenic sequence, segments orportions a gene with or without intergenic sequence, or sequence ofnucleic acids to which probes or primers are designed. Target nucleicacids may include wild type sequences, nucleic acid sequences containingmutations, deletions or duplications, tandem repeat regions, a gene ofinterest, a region of a gene of interest or any upstream or downstreamregion thereof. Target nucleic acids may represent alternative sequencesor alleles of a particular gene. Target nucleic acids may be derivedfrom genomic DNA, cDNA, or RNA. As used herein, target nucleic acid maybe native DNA or a PCR-amplified product. In one embodiment, the targetnucleic acid is a fragment of a chromosomal inversion involving a fusionof the EML4-ALK genes.

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds, under which nucleic acid hybridizations are conducted. Withhigh stringency conditions, nucleic acid base pairing will occur onlybetween nucleic acids that have sufficiently long segments with a highfrequency of complementary base sequences. Exemplary hybridizationconditions are as follows. High stringency generally refers toconditions that permit hybridization of only those nucleic acidsequences that form stable hybrids in 0.018M NaCl at 65° C. Highstringency conditions can be provided, for example, by hybridization in50% formamide, 5×Denhardt's solution, 5×SSC (saline sodium citrate) 0.2%SDS (sodium dodecyl sulphate) at 42° C., followed by washing in 0.1×SSC,and 0.1% SDS at 65° C. Moderate stringency refers to conditionsequivalent to hybridization in 50% formamide, 5×Denhardt's solution,5×SSC, 0.2% SDS at 42° C., followed by washing in 0.2×SSC, 0.2% SDS, at65° C. Low stringency refers to conditions equivalent to hybridizationin 10% formamide, 5×Denhardt's solution, 6×SSC, 0.2% SDS, followed bywashing in 1×SSC, 0.2% SDS, at 50° C.

As used herein the term “substantially identical” refers to apolypeptide or nucleic acid exhibiting at least 50%, 75%, 85%, 90%, 95%,or even 99% identity to a reference amino acid or nucleic acid sequenceover the region of comparison. For polypeptides, the length ofcomparison sequences will generally be at least 20, 30, 40, or 50 aminoacids or more, or the full length of the polypeptide. For nucleic acids,the length of comparison sequences will generally be at least 10, 15,20, 25, 30, 40, 50, 75, or 100 nucleotides or more, or the full lengthof the nucleic acid.

As used herein, the term “patient” refers to a subject who receivesmedical care, attention or treatment. As used herein, the term is meantto encompass a person having or suspected of having a disease includinga person who may be symptomatic for a disease but who has not yet beendiagnosed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a chromatogram showing the results of amplifying RNA fromFFPE tissue from a NSCLC patient using an unlableled EML4 exon 17forward primer and FAM-labeled ALK exon 20 reverse primer. Two peakswere observed at position 171 bp and 238 bp, indicating presence of atleast two fusion variants. FIG. 1B and FIG. 1C are electropherogramsfrom reverse sequencing reactions. The 171 bp peak (FIG. 1B) consistedof a complete EML4 exon 17 and ALK exon 20 separated by 30 bp ofadjacent ALK intron 19-20 sequence and the 238 bp peak (FIG. 1C) alsocontains a complete EML4 exon 17 and ALK exon 20 and adjacent ALK intron19-20 with an added 4 bp (boxed) of that intron and furthermore includes61 bp of non-adjacent intron 17-18 separating EML4 exon 17 and ALKintron sequence.

FIG. 2A and FIG. 2B are schematic diagrams of fusion transcriptsresulting from EML4 (light) and ALK (dark) genetic rearrangement of theEML4-ALK variants 8a and 8b, respectively. Intronic sequences carriedover to the fusion transcripts are indicated by dotted lines. PutativeEML4 truncation containing the N-terminal region of EML4 consisting ofHELP and partial WD repeat domains (variant 8a) and fusion of theN-terminal region of EML4 to the C-terminal region of ALK containing theprotein tyrosine kinase domain (variant 8b). Breakpoints indicated witha dotted line. IDS, intron-derived sequence; TM, transmembrane domain.

DETAILED DESCRIPTION

In accordance with the present invention, methods are provided fordetecting a particular nucleic acid segment of interest in a sample ofnucleic acids. In particular embodiments, the nucleic acid segment ofinterest includes the junction of a fusion of the EML4-ALK genes. Thisinformation may be used to determine if an individual is suffering fromor is susceptible to a neoplastic disorder, such as NSCLC.

In practicing the methods described herein, many conventional techniquesin molecular biology, protein biochemistry, cell biology, immunology,microbiology and recombinant DNA are used. These techniques arewell-known and are explained in, e.g., Current Protocols in MolecularBiology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al., MolecularCloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989); DNA Cloning: A PracticalApproach, Vols. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis,Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds.(1985); Transcription and Translation, Hames & Higgins, Eds. (1984);Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes(IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; theseries, Meth. Enzymol., (Academic Press, Inc., 1984); Gene TransferVectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring HarborLaboratory, N Y, 1987); and Meth. Enzymol., Vols. 154 and 155, Wu &Grossman, and Wu, Eds., respectively.

Assays for the Detection of EML4-ALK Fusion Genes

In one aspect, the present methods are designed to detect various targetnucleic acids associated with cancer, e.g., non-small cell lung cancer.In one embodiment, an assay for an EML4-ALK gene fusion involvesdetecting nucleic acid segments corresponding to the fusion gene thatare relevant to a diagnosis of cancer. Nucleic acid segments may bedetected in a variety of ways, which are described in further detailbelow. In one embodiment, an assay for EML4-ALK gene fusions may beperformed using PCR or RT-PCR. In another embodiment, an assay for anEML4-ALK gene fusion involves detecting an aberrant protein.

In one embodiment, the EML4-ALK variant (designated “variant 8a”) is afusion between exon 20 of ALK and exon 17 of EML4 with an insertion of30 nucleotides of ALK introns 19-20. According to the nomenclature ofHorn and Pao, J Clin Oncol 27: 4232-4235 (2009), this variant would benamed “E17;ins30A20”. The sequence of variant 8a surrounding thebreakpoint is set forth in SEQ ID NO: 1. Nucleotides 1-24 of SEQ ID NO:1 represent the 3′-region of ALK exon 20. Nucleotides 55-78 of SEQ IDNO: 1 represent the 5′-region of EML4 exon 17. The underlined region(nucleotides 25-54) of SEQ ID NO: 1 is the 30 nucleotide insert from ALKintrons 19-20.

(SEQ ID NO: 1) GCTCCTGGTG CTTCCGGCGG TACACTGCAG GTGGGTGGTCAGCTGCAACA TGGCCTGCCT GAGTGCGTTC CTATGGCC

Thus, variant 8a may be detected by detecting the presence of the 5′insertion junction (CGGCGGTACA:CTGCAGGTGG; SEQ ID NO: 26), the3′-insertion junction (GCAACATGGC:CTGCCTGAGT; SEQ ID NO: 27), or theentire insertion (underlined), wherein the colon indicates the junctionbetween the intron 19-20 insertion and the exonic sequences. Thepresence of the 5′- and 3′-junctions (e.g., SEQ ID NOs: 26 and 27) orthe full ALK intron 19-20 insertion may be assessed by any convenientmethod including nucleotide sequence and/or hybridization of anoligonucleotide probe that contains a sufficient number of nucleotideson either side of the junction in order that specific hybridization canbe assessed. The simultaneous presence of both the 5′- and 3′-junctions(SEQ ID NOs: 26 and 27) in a single sample, or the ALK intron 19-20insert alone (e.g., in mRNA) are indicative of variant 8a EML4-ALKtranslocation. The exemplary 20-mer sequences provided above, orcomplements thereof, may be used hybridization probes or otherwise maybe used as target regions of interest for assessment of variant 8a.

In one embodiment, an EML4-ALK variant (designated “variant 8b”) is afusion between exon 20 of ALK and exon 17 of EML4 with an insertion of95 nucleotides between the ALK and EML4 exons. The insertion sequence,in the 5′-to-3′ direction consists of the same 30 nucleotides from ALKintrons 19-20 as in variant 8a, a four nucleotide insert (CTGG), and 61nucleotides from EML4 introns 17-18. According to the nomenclature ofHorn and Pao, this variant would be named “E17ins30;ins65A20”. Thesequence of variant 8b surrounding the breakpoint is set forth in SEQ IDNO: 2. Nucleotides 1-24 of SEQ ID NO: 2 represent the 3′-region of ALKexon 20. Nucleotides 120-143 of SEQ ID NO: 2 represent the 5′-region ofEML4 exon 17. The underlined region (nucleotides 25-54) of SEQ ID NO: 2is the 30 nucleotide insert from ALK introns 19-20. The lower casenucleotides (nucleotides 55-58) is the four base insert. And, the bold,italicized nucleotides (nucleotides 59-119) is the inserted sequencefrom EML4 introns 17-18.

(SEQ ID NO: 2) GCTCCTGGTG CTTCCGGCGG TACACTGCAG GTGGGTGGTCAGCTGCAACA TGGCctgg

 

 

 

 

 

C TGCCTGAGTG CGTTCCTATG GCC

Thus, variant 8b may be detected by detecting the present of the 5′insertion junction (e.g., SEQ ID NO: 26), the 3′-insertion junction(TTTTTGTCTC:CTGCCTGAGT; SEQ ID NO: 28), or the entire 95 bp insertion,wherein the colon indicates the junction between the intronic insertionsand the exonic sequences. The presence of the 5′- and 3′-junctions(e.g., SEQ ID NOs: 26 and 28) or the full 95 bp insertion (or adiagnostic portion thereof) may be assessed by any convenient methodincluding nucleotide sequence and/or hybridization of an oligonucleotideprobe that contains a sufficient number of nucleotides on either side ofthe junction in order that specific hybridization can be assessed. Thesimultaneous presence of both the 5′- and 3′-junctions (SEQ ID NOs: 26and 28) in a single sample, or the full 95 bp insertion (or a diagnosticportion thereof) (e.g., in mRNA) are indicative of variant 8b EML4-ALKtranslocation. The exemplary 20-mer sequences provided above, orcomplements thereof, may be used hybridization probes or otherwise maybe used as target regions of interest for assessment of variant 8b.

In one aspect, the methods may detect two or more EML-ALK gene fusionvariants, e.g., in a multiplex or multiple singleplex PCR assays. Forinstance the assay may detect variant 8a and/or variant 8b with one ormore of the following variants: variant 1, variant 2, variant 3a,variant 3b, variant 4, variant 5a, variant 5b, variant 6, and variant 6.A description of these variants is set forth in Table 1, and aredescribed further in Horn and Pao, J Clin Oncol 27: 4232-4235 (2009).

TABLE 1 EML4-ALK Fusion Variants Variant Description V1 (E13; A20)Variant 1 fuses exon 13 of EML4 to exon 20 of ALK. V2 (E20; A20) Variant2 fuses exon 20 of EML4 to exon 20 of ALK. V3a (E6a; A20) Variant 3afuses exon 6a of EML4 to exon 20 of ALK. V3b (E6b; A20) Variant 3b fusesexon 6b of EML4 to exon 20 of ALK. V4 (E14; ins11del49A20) Variant 4fuses exon 14 of EML4 with an additional 11 nucleotides of unknownorigin to nucleotide 50 within exon 20 ALK. V5a (E2; A20) Variant 5afuses exon 2 of EML4 to exon 20 of ALK. V5b (E2; ins117A20) Variant 5bfuses exon 2 of EML4 to intron 19 of ALK (including 117 nucleotides fromintron 19). V6 (E13; ins69A20) Variant 6 fuses exon 13 of EML4 to intron19 of ALK (including 69 nucleotides from intron 19). V7 (E14; del12A20)Variant 7 fuses exon 13 of EML4 to nucleotide 13 in within exon 20 ofALK. V8a (E17; ins30A20) Variant 8a fuses exon 17 of EML4 to intron 19of ALK (including 35 nucleotides from intron 19). V8b (E17ins30;ins65A20) Variant 8b fuses exon 17 (with an addition 60 nucleotides fromintron 18) of EML4 to intron 19 of ALK (including 35 nucleotides fromintron 19).

Exemplary RT-PCR primers for the detection of EML-ALK fusions are shownin Table 2 below. With regard to the exemplary primers and probes, thoseskilled in the art will readily recognize that nucleic acid moleculesmay be double-stranded molecules and that reference to a particular siteon one strand refers, as well, to the corresponding site on acomplementary strand. In defining a variant position, allele, ornucleotide sequence, reference to an adenine, a thymine (uridine), acytosine, or a guanine at a particular site on one strand of a nucleicacid molecule also defines the thymine (uridine), adenine, guanine, orcytosine (respectively) at the corresponding site on a complementarystrand of the nucleic acid molecule. Thus, reference may be made toeither strand in order to refer to a particular variant position,allele, or nucleotide sequence. Probes and primers, may be designed tohybridize to either strand and detection methods disclosed herein maygenerally target either strand.

Sample Collection and Preparation

The methods and compositions of this invention may be used to detectnucleic acids associated with various EML4-ALK gene fusions using abiological sample obtained from an individual. The nucleic acid (DNA orRNA) may be isolated from the sample according to any methods well knownto those of skill in the art. Biological samples may be obtained bystandard procedures and may be used immediately or stored, underconditions appropriate for the type of biological sample, for later use.

Starting material for the detection assays is typically a clinicalsample, which is suspected to contain the target nucleic acids. Anexample of a clinical sample is a tissue from a biopsy. Next, thenucleic acids may be separated from proteins and sugars present in theoriginal sample. Any purification methods known in the art may be usedin the context of the present invention. Nucleic acid sequences in thesample can successfully be amplified using in vitro amplification, suchas PCR. Typically, any compounds that may inhibit polymerases areremoved from the nucleic acids.

Methods of obtaining test samples are well known to those of skill inthe art and include, but are not limited to, aspirations, tissuesections, swabs, drawing of blood or other fluids, surgical or needlebiopsies, and the like. The test sample may be obtained from anindividual or patient. The test sample may contain cells, tissues orfluid obtained from a patient suspected being afflicted with or cancer,e.g., NSCLC. The test sample may be a cell-containing liquid or atissue. Samples may include, but are not limited to, cells from avaginal swab, amniotic fluid, biopsies, blood, blood cells, bone marrow,fine needle biopsy samples, peritoneal fluid, amniotic fluid, plasma,pleural fluid, saliva, semen, serum, tissue or tissue homogenates,frozen or paraffin sections of tissue. Samples may also be processed,such as sectioning of tissues, fractionation, purification, or cellularorganelle separation.

If necessary, the sample may be collected or concentrated bycentrifugation and the like. The cells of the sample may be subjected tolysis, such as by treatments with enzymes, heat, surfactants,ultrasonication, or a combination thereof. The lysis treatment isperformed in order to obtain a sufficient amount of nucleic acid derivedfrom the cells in the sample to detect using polymerase chain reaction.

Nucleic Acid Extraction and Amplification

The nucleic acid to be amplified may be from a biological sample such asa tissue sample and the like. Various methods of extraction are suitablefor isolating the DNA or RNA. Suitable methods include phenol andchloroform extraction. See Maniatis et al., Molecular Cloning, ALaboratory Manual, 2d, Cold Spring Harbor Laboratory Press, pp. 16-54(1989). Numerous commercial kits also yield suitable DNA and RNAincluding, but not limited to, QIAamp™ mini blood kit, AgencourtGenfind™, Roche Cobas® Roche MagNA Pure® or phenol:chloroform extractionusing Eppendorf Phase Lock Gels®, and the NucliSens extraction kit(Biomerieux, Marcy l'Etoile, France).

Nucleic acid extracted from cells or tissues can be amplified usingnucleic acid amplification techniques well know in the art. By way ofexample, but not by way of limitation, these techniques can include thepolymerase chain reaction (PCR), reverse transcriptase polymerase chainreaction (RT-PCR), nested PCR, ligase chain reaction. See Abravaya, K.,et al., Nucleic Acids Research, 23:675-682, (1995), branched DNA signalamplification, Urdea, M. S., et al., AIDS, 7 (suppl 2):S11-S 14, (1993),amplifiable RNA reporters, Q-beta replication, transcription-basedamplification, boomerang DNA amplification, strand displacementactivation, cycling probe technology, isothermal nucleic acid sequencebased amplification (NASBA). See Kievits, T. et al., J VirologicalMethods, 35:273-286, (1991), Invader Technology, or other sequencereplication assays or signal amplification assays may also be used.These methods of amplification are each described briefly below and arewell-known in the art.

Some methods employ reverse transcription of RNA to cDNA. The method ofreverse transcription and amplification may be performed by previouslypublished or recommended procedures. Various reverse transcriptases maybe used, including, but not limited to, MMLV RT, RNase H mutants of MMLVRT such as Superscript and Superscript II (Life Technologies, GIBCO BRL,Gaithersburg, Md.), AMV RT, and thermostable reverse transcriptase fromThermus thermophilus. For example, one method which may be used toconvert RNA to cDNA is the protocol adapted from the Superscript IIPreamplification system (Life Technologies, GIBCO BRL, Gaithersburg,Md.; catalog no. 18089-011), as described by Rashtchian, A., PCR MethodsApplic., 4:S83-S91, (1994).

LCR is a method of DNA amplification similar to PCR, except that it usesfour primers instead of two and uses the enzyme ligase to ligate or jointwo segments of DNA. LCR can be performed as according to Moore et al.,J Clin Micro, 36(4):1028-1031 (1998). Briefly, an LCR reaction mixturecontains two pair of primers, dNTP, DNA ligase and DNA polymeraserepresenting about 90 μl, to which is added 100 μl of isolated nucleicacid from the target organism. Amplification is performed in a thermalcycler (e.g., LCx of Abbott Labs, Chicago, Ill.).

TAS is a system of nucleic acid amplification in which each cycle iscomprised of a cDNA synthesis step and an RNA transcription step. In thecDNA synthesis step, a sequence recognized by a DNA-dependent RNApolymerase (i.e., a polymerase-binding sequence or PBS) is inserted intothe cDNA copy downstream of the target or marker sequence to beamplified using a two-domain oligonucleotide primer. In the second step,an RNA polymerase is used to synthesize multiple copies of RNA from thecDNA template. Amplification using TAS requires only a few cyclesbecause DNA-dependent RNA transcription can result in 10-1000 copies foreach copy of cDNA template. TAS can be performed according to Kwoh etal., PNAS, 86:1173-7 (1989). Briefly, extracted RNA is combined with TASamplification buffer and bovine serum albumin, dNTPs, NTPs, and twooligonucleotide primers, one of which contains a PBS. The sample isheated to denature the RNA template and cooled to the primer annealingtemperature. Reverse transcriptase (RT) is added the sample incubated atthe appropriate temperature to allow cDNA elongation. Subsequently T7RNA polymerase is added and the sample is incubated at 37° C. forapproximately 25 min for the synthesis of RNA. The above steps are thenrepeated. Alternatively, after the initial cDNA synthesis, both RT andRNA polymerase are added following a 1 min 100° C. denaturation followedby an RNA elongation of approximately 30 min at 37° C. TAS can be alsobe performed on solid phase as according to Wylie et al., J Clin Micro,36(12):3488-3491 (1998). In this method, nucleic acid targets arecaptured with magnetic beads containing specific capture primers. Thebeads with captured targets are washed and pelleted before addingamplification reagents which contains amplification primers, dNTP, NTP,2500 U of reverse transcriptase and 2500 U of T7 RNA polymerase. A 100μl TMA reaction mixture is placed in a tube, 200 μl oil reagent is addedand amplification is accomplished by incubation at 42° C. in a waterbathfor one hour.

NASBA is a transcription-based amplification method which amplifies RNAfrom either an RNA or DNA target. NASBA is a method used for thecontinuous amplification of nucleic acids in a single mixture at onetemperature. For example, for RNA amplification, avian myeloblastosisvirus (AMV) reverse transcriptase, RNase H and T7 RNA polymerase areused. This method can be performed as according to Heim, et al., NucleicAcids Res., 26(9):2250-2251 (1998). Briefly, an NASBA reaction mixturecontains two specific primers, dNTP, NTP, 6.4 U of AMV reversetranscriptase, 0.08 U of E. coli Rnase H, and 32 U of T7 RNA polymerase.The amplification is carried out for 120 min at 41° C. in a total volumeof 20 μl.

In a related method, self-sustained sequence-replication (3SR) reaction,isothermal amplification of target DNA or RNA sequences in vitro usingthree enzymatic activities: reverse transcriptase, DNA-dependent RNApolymerase and E. coli ribonuclease H. This method may be modified froma 3-enzyme system to a 2-enzyme system by using human immunodeficiencyvirus (HIV)-1 reverse transcriptase instead of avian myeloblastosisvirus (AMV) reverse transcriptase to allow amplification with T7 RNApolymerase but without E. coli ribonuclease H. In the 2-enzyme 3SR, theamplified RNA is obtained in a purer form compared with the 3-enzyme 3SR(Gebinoga & Oehlenschlager Eur J Biochem, 235:256-261, 1996).

SDA is an isothermal nucleic acid amplification method. A primercontaining a restriction site is annealed to the template. Amplificationprimers are then annealed to 5′ adjacent sequences (forming a nick) andamplification is started at a fixed temperature. Newly synthesized DNAstrands are nicked by a restriction enzyme and the polymeraseamplification begins again, displacing the newly synthesized strands.SDA can be performed as according to Walker, et al., PNAS, 89:392-6(1992). Briefly, an SDA reaction mixture contains four SDA primers,dGTP, dCTP, TTP, dATP, 150 U of Hinc II, and 5 U ofexonuclease-deficient of the large fragment of E. coli DNA polymerase I(exo⁻ Klenow polymerase). The sample mixture is heated 95° C. for 4 minto denature target DNA prior to addition of the enzymes. After additionof the two enzymes, amplification is carried out for 120 min at 37° C.in a total volume of 50 μl. Then, the reaction is terminated by heatingfor 2 min at 95° C.

The Q-beta replication system uses RNA as a template. Q-beta replicasesynthesizes the single-stranded RNA genome of the coliphage Qβ. Cleavingthe RNA and ligating in a nucleic acid of interest allows thereplication of that sequence when the RNA is replicated by Q-betareplicase (Kramer & Lizardi, Trends Biotechnol., 1991 9(2):53-8, 1991).

In suitable embodiments, PCR is used to amplify a target sequence ofinterest. PCR is a technique for making many copies of a specifictemplate DNA sequence. The reaction consists of multiple amplificationcycles and is initiated using a pair of primer sequences that hybridizeto the 5′ and 3′ ends of the sequence to be copied. The amplificationcycle includes an initial denaturation, and typically up to 50 cycles ofannealing, strand elongation and strand separation (denaturation). Ineach cycle of the reaction, the DNA sequence between the primers iscopied. Primers can bind to the copied DNA as well as the originaltemplate sequence, so the total number of copies increases exponentiallywith time. PCR can be performed as according to Whelan et al., J of ClinMicro, 33(3):556-561 (1995). Briefly, a PCR reaction mixture includestwo specific primers, dNTPs, approximately 0.25 U of Taq polymerase, and1×PCR Buffer.

The skilled artisan is capable of designing and preparing primers thatare appropriate for amplifying a target or marker sequence. The lengthof the amplification primers depends on several factors including thenucleotide sequence identity and the temperature at which these nucleicacids are hybridized or used during in vitro nucleic acid amplification.The considerations necessary to determine a preferred length for anamplification primer of a particular sequence identity are well-known toa person of ordinary skill. For example, the length of a short nucleicacid or oligonucleotide can relate to its hybridization specificity orselectivity. Exemplary primers for detecting EML4-ALK gene fusion byRT-PLR are set forth in Table 2.

In some embodiments, the amplification may include a labeled primer orprobe, thereby allowing detection of the amplification productscorresponding to that primer or probe. In particular embodiments, theamplification may include a multiplicity of labeled primers or probes;such primers may be distinguishably labeled, allowing the simultaneousdetection of multiple amplification products.

In one embodiment, a primer or probe is labeled with a fluorogenicreporter dye that emits a detectable signal. While a suitable reporterdye is a fluorescent dye, any reporter dye that can be attached to adetection reagent such as an oligonucleotide probe or primer is suitablefor use in the invention. Such dyes include, but are not limited to,Acridine, AMCA, BODIPY, Cascade Blue, Cy2, Cy3, Cy5, Cy7, Edans, Eosin,Erythrosin, Fluorescein, 6-Fam, Tet, Joe, Hex, Oregon Green, Rhodamine,Rhodol Green, Tamra, Rox, and Texas Red.

In yet another embodiment, the detection reagent may be further labeledwith a quencher dye such as Tamra, Dabcyl, or Black Hole Quencher®(BHQ), especially when the reagent is used as a self-quenching probesuch as a TaqMan® (U.S. Pat. Nos. 5,210,015 and 5,538,848) or MolecularBeacon probe (U.S. Pat. Nos. 5,118,801 and 5,312,728), or other stemlessor linear beacon probe (Livak et al., 1995, PCR Method Appl., 4:357-362;Tyagi et al, 1996, Nature Biotechnology, 14:303-308; Nazarenko et al.,1997, Nucl. Acids Res., 25:2516-2521; U.S. Pat. Nos. 5,866,336 and6,117,635).

Nucleic acids may be amplified prior to detection or may be detecteddirectly during an amplification step (i.e., “real-time” methods). Insome embodiments, the target sequence is amplified using a labeledprimer such that the resulting amplicon is detectably labeled. In someembodiments, the primer is fluorescently labeled. In some embodiments,the target sequence is amplified and the resulting amplicon is detectedby electrophoresis.

In one embodiment, detection of a target nucleic acid, such as a nucleicacid from an EML4-ALK gene fusion, is performed using the TaqMan® assay,which is also known as the 5′ nuclease assay (U.S. Pat. Nos. 5,210,015and 5,538,848). The TaqMan® assay detects the accumulation of a specificamplified product during PCR. The TaqMan® assay utilizes anoligonucleotide probe labeled with a fluorescent reporter dye and aquencher dye. The reporter dye is excited by irradiation at anappropriate wavelength, it transfers energy to the quencher dye in thesame probe via a process called fluorescence resonance energy transfer(FRET). When attached to the probe, the excited reporter dye does notemit a signal. The proximity of the quencher dye to the reporter dye inthe intact probe maintains a reduced fluorescence for the reporter. Thereporter dye and quencher dye may be at the 5′ most and the 3′ mostends, respectively or vice versa. Alternatively, the reporter dye may beat the 5′ or 3′ most end while the quencher dye is attached to aninternal nucleotide, or vice versa. In yet another embodiment, both thereporter and the quencher may be attached to internal nucleotides at adistance from each other such that fluorescence of the reporter isreduced.

During PCR, the 5′ nuclease activity of DNA polymerase cleaves theprobe, thereby separating the reporter dye and the quencher dye andresulting in increased fluorescence of the reporter. Accumulation of PCRproduct is detected directly by monitoring the increase in fluorescenceof the reporter dye. The DNA polymerase cleaves the probe between thereporter dye and the quencher dye only if the probe hybridizes to thetarget-containing template which is amplified during PCR.

TaqMan® primer and probe sequences can readily be determined using thevariant and associated nucleic acid sequence information providedherein. A number of computer programs, such as Primer Express (AppliedBiosystems, Foster City, Calif.), can be used to rapidly obtain optimalprimer/probe sets. It will be apparent to one of skill in the art thatsuch primers and probes for detecting the target nucleic acids areuseful in diagnostic assays for neoplastic disorders, such as NSCLC, andcan be readily incorporated into a kit format. The present inventionalso includes modifications of the TaqMan® assay well known in the artsuch as the use of Molecular Beacon probes (U.S. Pat. Nos. 5,118,801 and5,312,728) and other variant formats (U.S. Pat. Nos. 5,866,336 and6,117,635).

In an illustrative embodiment, real time PCR is performed using TaqMan®probes in combination with a suitable amplification/analyzer such as theABI Prism® 7900HT Sequence Detection System. The ABI PRISM® 7900HTSequence Detection System is a high-throughput real-time PCR system thatdetects and quantitates nucleic acid sequences. Real time detection onthe ABI Prism 7900HT or 7900HT Sequence Detector monitors fluorescenceand calculates Rn during each PCR cycle. The threshold cycle, or Ctvalue, is the cycle at which fluorescence intersects the thresholdvalue. The threshold value is determined by the sequence detectionsystem software or manually. The Ct can be correlated to the initialamount of nucleic acids or number of starting cells using a standardcurve.

Other methods of probe hybridization detected in real time can be usedfor detecting amplification of a target or marker sequence flanking atandem repeat region. For example, the commercially available MGBEclipse™ probes (Epoch Biosciences), which do not rely on a probedegradation can be used. MGB Eclipse™ probes work by ahybridization-triggered fluorescence mechanism. MGB Eclipse™ probes havethe Eclipse™ Dark Quencher and the MGB positioned at the 5′-end of theprobe. The fluorophore is located on the 3′-end of the probe. When theprobe is in solution and not hybridized, the three dimensionalconformation brings the quencher into close proximity of thefluorophore, and the fluorescence is quenched. However, when the probeanneals to a target or marker sequence, the probe is unfolded, thequencher is moved from the fluorophore, and the resultant fluorescencecan be detected.

Oligonucleotide probes can be designed which are between about 10 andabout 100 nucleotides in length and hybridize to the amplified region.Oligonucleotides probes are preferably 12 to 70 nucleotides; morepreferably 15-60 nucleotides in length; and most preferably 15-25nucleotides in length. The probe may be labeled. Amplified fragments maybe detected using standard gel electrophoresis methods. For example, insome embodiments, amplified fractions are separated on an agarose geland stained with ethidium bromide by methods known in the art to detectamplified fragments.

As a quality control measure, an internal amplification control may beincluded in one or more samples to be extracted and amplified. Theskilled artisan will understand that any detectable sequence that is nottypically present in the sample can be used as the control sequence. Acontrol sequence can be produced synthetically. If PCR amplification issuccessful, the internal amplification control amplicons can then bedetected. Additionally, if included in the sample prior to purificationof nucleic acids, the control sequences can also act as a positivepurification control.

Protein Assays

In one aspect, the present invention provides methods of detecting amutant protein associated with a neoplastic disorder, such as NSCLC. Inone embodiment, the methods provide for detection of an EML4-ALK fusionprotein. The presence of EML4-ALK fusion proteins can be measured byimmunoassay, using antibodies specific for the mutant protein. Lack ofantibody binding would indicate the absence of mutant EML4-ALK moleculesand suggest that the subject does not have or is not susceptible to aneoplastic disorder associated with the mutant EML4-ALK protein.Antibodies specific to wild-type EML4 or ALK protein may be used as acontrol.

Antibodies which are specifically reactive with mutant EML4-ALK proteinsmay be obtained in a number of ways which will be readily apparent tothose skilled in the art. The fusion protein can be produced in arecombinant system using the mutant nucleotide sequence. The recombinantprotein can be injected into an animal as an immunogen to elicitpolyclonal antibody production. The resultant polyclonal antisera may beused directly or may be purified by, for example, affinity absorptionusing recombinantly produced EML4-ALK fusion proteins coupled to aninsoluble support.

In another alternative, monoclonal antibodies specificallyimmunoreactive with the fusion protein may be prepared according to wellknown methods (See, e.g., Kohler and Milstein, 1976, Eur. J. Immunol.,6:611), using a peptide as an immunogen, using it for selection or usingit for both functions. These and other methods for preparing antibodiesthat are specifically immunoreactive with the recombinant protein ofthis invention are easily within the skill of the ordinary worker in theart. Suitably, antibodies specific for mutant EML4-ALK fusion proteinwill not react with normal (wild type) EML4 or ALK proteins. Similarmethods can be used to produce antibodies specifically immunoreactivewith wild-type EML4 or ALK.

Kits

In a further aspect, the invention disclosure provides kits fordiagnosing a neoplastic disorder in an individual, the kit comprising: aset of reagents for determining the presence or absence, or differentialpresence, of EML4-ALK gene fusions. In one embodiment, at least oneprimer pair is selected from the group consisting of: SEQ ID NOs: 3/25,4/25, 5/25, 6/25, 7/25, 8/25, 9/25, 10/25, 11/25, 12/25, 13/25; 16/25,17/25, 18/25, 19/25, 20/25, 21/25, 22/25, 23/25, and 24/25. In oneembodiment, the kit contains one or more of the primers of SEQ ID NOs:3-28.

EXAMPLE

The present invention is further illustrated by the following examples,which should not be construed as limiting in any way.

Example 1: Cytogenetic Analysis

FFPE sections 4-5 μm in size were used for FISH with an ALK dual-colorbreak-apart probe (Abbott Molecular, Inc., Des Plaines, Ill.); theslides were deparaffinized prior to probe application. FISH analysis wasperformed using a Nikon 50i fluorescence microscope (Nikon Corp., Tokyo,Japan). The images were captured using a CCD camera and Isis® imagingsystem (MetaSystems Group, Inc., Watertown, Mass.). Unstained slideswere deparaffinized and stained with CONFIRM™ anti-ALK1 primary antibody(mouse monoclonal clone ALK01; Ventana Medical Systems, Tucson Ariz.).All immunohistochemistry steps were performed using the BenchMark XT,according to manufacturer's protocol (Ventana Medical Systems, Tucson,Ariz.).

Cytogenetic analysis of a non-small cell lung cancer (NSCLC) patientrevealed breakage within the ALK gene. Both haploid and diploid cellswere observed where split signal from the 5′ and 3′ ALK probes wereevident. Likewise, immunohistochemistry of the tissue sample confirmedsignificant ALK1 staining indicating overexpression of ALK1 in themalignant cells.

Example 2: Evaluation of EML4-ALK Fusions

Because the FISH and IHC results from Example 1 indicated an oncogenicALK1 rearrangement, the molecular characteristics were furtherinvestigated under the assumption that there was an EML4-ALK fusionevent. RT-PCR amplification with primers to detect the first 11published EML4-ALK fusion variants (variant 1, 2, 3a, 3b, 4, 5a, 5b, 6,and 7) yielded results that suggested the likely involvement of apreviously undescribed EML4-ALK fusion variant(s).

Methods.

Sixty (60) patient samples were analyzed with the multiplex assay: 55formalin-fixed paraffin-embedded (FFPE) NSCLC samples (37 patients withadenocarcinoma, 11 squamous cell carcinoma, 5 adenosquamous cellcarcinoma and 2 large cell carcinomas), 4 cell line samples (3 NSCLC; 1Prostate Cancer), and 1 control RNA (Human Total RNA, AppliedBiosystems). Tissue blocks were sectioned onto slides for H&E stainingor unstained for RNA extraction. Tumor area was identified by a licensedpathologist and tissue from this area was scraped for RNA extractionwith HighPure miRNA Isolation kit (Roche Applied Science, Mannheim,Germany). RNA was DNase I digested with DNA-free (AppliedBiosystems/Ambion, Austin Tex.) prior to amplification.

After RNA extraction, RT-PCR was performed using the RNA UltraSense™One-Step qRT-PCR System (Invitrogen, Carlsbad, Calif.). 23 primers (22unlabeled EML4 forward; 1 FAM labeled ALK reverse) were included in 4master mixes to amplify EML4-ALK fusions initiating within the first 22EML4 exons to ALK exon 20 (Table 2). One endogenous control(beta-2-microglobulin) primer set was included in a separate reaction.Thermocycling was performed as follows: RNA reverse transcribed byincubation at 55° C. for 30 min followed by denaturing step at 94° C.for 2 min; PCR amplification performed by 40 cycles of denaturation at94° C. for 15 sec; annealing at 60° C. for 30 min; extension at 68° C.;and final extension at 68° C. for 5 min following cycling. RT-PCR toquery alternative splicing in intron 17 was performed as above, withexception that the annealing temperature was 50° C. Exemplary primersinclude:

TABLE 2 Exemplary RT-PCR Primers for Detecting EML4-ALK Gene FusionsPrimer Target Sequence (5′ to 3′) SEQ ID NO: EML4 Exon 1 (Forward)CGGTCCGCTGAATGAAGT SEQ ID NO: 3 EML4 Exon 2 (Forward)AAGATCATGTGGCCTCAGTG SEQ ID NO: 4 EML4 Exon 3 (Forward)TGGTGCAAACAGAAAACCAA SEQ ID NO: 5 EML4 Exon 4 (Forward)CCCTCTTCACAACCTCTCCA SEQ ID NO: 6 EML4 Exon 5 (Forward)ACGACCATCACCAGCTGAAA SEQ ID NO: 7 EML4 Exon 6 (Forward)CTGCAGACAAGCATAAAGATG SEQ ID NO: 8 EML4 Exon 7 (Forward)GTCGGCCAATTACCATGTTC SEQ ID NO: 9 EML4 Exon 8 (Forward)CTTCCGACCGGGAAAATAGT SEQ ID NO: 10 EML4 Exon 9 (Forward)ACATCCTGACAAAATTAGGATTGC SEQ ID NO: 11 EML4 Exon 10 (Forward)CCTCTACAACCCCACGTCAG SEQ ID NO: 12 EML4 Exon 11 (Forward)GCATATGCTTACTGTATGGGACTG SEQ ID NO: 13 EML4 Exon 12 (Forward)TTTCACCCAACAGATGCAAA SEQ ID NO: 14 EML4 Exon 13 (Forward)GACTCAGGTGGAGTCATGC SEQ ID NO: 15 EML4 Exon 14 (Forward)AAGCTCATGATGGCAGTGTG SEQ ID NO: 16 EML4 Exon 15 (Forward)TGTAGCAGAAGGAAAGGCAGA SEQ ID NO: 17 EML4 Exon 16 (Forward)GTCTTGCCACACATCCCTTC SEQ ID NO: 18 EML4 Exon 17 (Forward)CCAGGACACTGTGCAGATTT SEQ ID NO: 19 EML4 Exon 18 (Forward)AGGTGGTTTGTTCTGGATGC SEQ ID NO: 20 EML4 Exon 19 (Forward)CCTTCCTGGCTGTAGGATCTC SEQ ID NO: 21 EML4 Exon 20 (Forward)CAGATATGGAAGGTGCACTG SEQ ID NO: 22 EML4 Exon 21 (Forward)ATTCCAAATGGCTGCAAACT SEQ ID NO: 23 EML4 Exon 22 (Forward)AGCTGTTGCCGATGACTTTT SEQ ID NO: 24 ALK Exon 20 (Reverse)AGCTTGCTCAGCTTGTACTC SEQ ID NO: 25

RT-PCR products were diluted 1:10 with H₂O, denatured in formamidecontaining ROX GeneScan 350 size marker (Applied Biosystems, Foster CityCalif.), and size fractionated by capillary electrophoresis in an ABI3730 Genetic Analyzer (Applied Biosystems, Foster City Calif.). Resultswere analyzed by GeneMapper Software (Applied Biosystems, Foster CityCalif.). Samples with positive results were further analyzed bysingleplex RT-PCR to confirm exon involvement and novel fusions wereconfirmed by direct sequencing.

For cDNA sequencing, RT-PCR products were separated by capillaryelectrophoresis on a 2% agarose gel. Individual bands were cut out ofthe gel and DNA extracted by MinElute Gel Extraction Kit, according tomanufacturer's instructions (Qiagen, Valencia Calif.). Forward andreverse primers used in RT-PCR served as forward and reverse primers forsequencing using ABI Prism Big Dye Terminator v3.1 Cycle Sequencing Kit,according to manufacturer's instructions (Applied Biosystems, FosterCity Calif.).

For immunohistochemistry, unstained slides were deparaffinized andstained with CONFIRM™ anti-ALK1 primary antibody (mouse monoclonal cloneALK01; Ventana Medical Systems, Tucson Ariz.). All immunohistochemistrysteps were performed using the BenchMark XT, according to manufacturer'sprotocol (Ventana Medical Systems, Tucson Ariz.).

Results:

A fluorescent RT-PCR assay was designed utilizing a single FAM-labeledALK exon 20 reverse primer and 22 forward primers for each of the first22 exons of EML4 (Table 2). The forward primers were split into 4separate reactions, all containing the labeled ALK exon 20 reverseprimer. Upon fragment analysis, each known EML4-ALK fusion variant wouldyield a specific size that is utilized to identify the fusion(s)present. Furthermore, potential exon fusions would yield a unique sizeproduct for ease of identification of EML4 exon involvement, assuming noinsertions or deletions are present (Table 3).

TABLE 3 Expected amplicon size by variant EML4 exon Multiplex EML4/ALKvariant involvement Reaction# Amplicon Size (bp) Published EML4-ALKfusion variants 2, 3a, 3b, 4, 7, E20; ins18A20 20, 6, 14, 20 1 183, 112,145, 163, 187, 201 “V5” 18 2 167 1, 6, E17; ins68A20 13, 17 3 146, 215,144 “V4”, 5a, 5b 15, 2 4 134, 118, 235 Potential EML4 exon involvement*Unknown 1, 5, 9, 12, 16, 21 2 152, 199, 137, 181, 186, 170 Unknown 3, 7,10, 3 154, 170, 187, Unknown 4, 8, 11, 19, 22 4 144, 183, 131, 168, 137*Amplicon sizes are estimated based on no insertions or deletions

Screening of 55 NSCLC formalin-fixed paraffin embedded (FFPE) tumortissue samples resulted in detection of 9.1% (5/55) EML4-ALK fusionpositive cases, all of which were classified as adenocarcinoma (Table4). Four of the positive cases harbored previously described fusionvariants, the majority of which were variants 3a and/or 3b (3/5) and onevariant 1 (Table 5).

TABLE 4 Distrubution of EML4-ALK positive tumors by NSCLC subtype.Subtype n EML4-ALK Positive Total 55  9.1% (5/55) Adenocarcinoma 3713.5% (5/37) Squamous cell 11 0% Adenosquamous 5 0% Large cell carcinoma2 0%

TABLE 5 Distribution of EML4-ALK fusion variants. EML4-ALK PositiveVariant Reference Total 9.1% (5/55) See below Variant 1 1.8% (1/55) Sodaet al., 2007 Variant 2 0% Soda et al., 2007 Variant 3a 5.3% (3/55) Choiet al, 2008 Variant 3b 3.5% (2/55) Choi et al., 2008 Variant 4 0%Takeuchi et al., 2008 Variant “4” 0% Koivunen et al., 2008 Variant 5a 0%Takeuchi et al., 2008 Variant 5b 0% Takeuchi et al., 2008 Variant “5” 0%Wong et al., 2009 Variant 6 0% Takeuchi et al., 2009 Variant 7 0%Takeuchi et al., 2009 Variant 8a 1.8% (1/55) Present study Variant 8b1.8% (1/55) Present study Variant E17 0% Tokahashi et al., 2009 VariantE20 0% Tokahashi et al., 2009

In addition, one case yielded 2 strong amplification peaks at unexpectedsizes in the reaction containing EML4 exon 3, 7, 10, 13, and 17 (mastermix #3). In order to identify the EML4 exon involvement, separation ofthe primers into individual reactions revealed that both peaks resultedfrom amplification with the EML4 exon 17 forward primer yielding 2amplicons of 170 bp and 237 bp (FIG. 1A). ALK rearrangement was alsoconfirmed by fluorescence in situ hybridization (FISH) using break apartprobes to detect rearrangements at the 2p23 locus. Both haploid anddiploid cells were observed in the specimen harboring novel variants8a/b, where split signal from the 5′ and 3′ ALK probes were evident(data not shown).

Twenty-two additional specimens (3 more EML4-ALK positive and 19negative by RT-PCR) were also confirmed by FISH as described above for atotal of 23 of the 55 specimens (data not shown). Due to limited sample,FISH was not performed on the variant 1 positive specimen. All specimensthat tested negative by RT-PCR also tested negative by FISH. Three ofthe four total RT-PCR positives tested were also positive by FISH. Onesample tested positive for EML4-ALK fusion variants 3a and 3b by RT-PCR,but negative for by FISH. Upon repeat RNA extraction and RT-PCR,detection of variant 3a and 3b in this specimen by RT-PCR wasduplicated. These results suggest higher sensitivity of the RT-PCRassay.

Sequencing of the un-identified amplicons described above demonstratedthat both amplicons were composed of complete EML4 exon 17 and ALK exon20, differing in size based on varying partial intron insertions. The170 bp peak (variant 8a) consisted of EML4 exon 17 fused to ALK exon 20containing a 30 nucleotide intron 19 insertion, E17;ins30A20 (FIG. 1B).The 237 bp peak (variant 8b) consisted of EML4 exon 17, with aninsertion of 61 non-adjacent nucleotides from intron 17, fused to ALKexon 20 with a 34 nucleotide insertion from intron 19, E17ins61;ins34A20(FIG. 1C). As a result, the two fusion products formed likely consist ofEML4 exon 1-17 and ALK 20-29 with a 30 bp (8a) or 95 bp (8b) insertion(FIG. 2A).

In order to determine whether the EML4 intron 17 segment may be observedadjacent to exon 17 as a result of alternative splicing in normal EML4transcripts, RT-PCR was performed on 3 NSCLC cell lines (NCI-H2228,NCI-1299, NCI-H838), two normal lung cancer tissues and the variant 8a/bpositive lung cancer tissue using primers specific to exon 17 and the 61bp intron 17 segment. The only specimen that resulted in an amplicon ofexpected size was the variant 8a/b positive lung cancer specimen (datanot shown). This suggests that as a result of this specific paracentricinversion, a new alternative splice site is created in the pre-mRNAtranscript.

Based on the deduced amino acid sequence, the translated variant 8aprotein yields a 660 a.a. protein (SEQ ID NO: 29) and variant 8b yieldsa 1250 a.a. protein (SEQ ID NO: 30), shown below. Fusion variant 8aappears to encode an EML4 truncation with no functional ALK domainsresulting from the presence of an early stop codon located in the 30 bpinsertion (FIG. 2B). However, variant 8b contains an in-frame 95 bpinsertion resulting in the presence of a putatively functional ALKprotein tyrosine kinase domain (underlined amino acids in SEQ ID NO: 30below). (See FIG. 2B).

(SEQ ID NO: 29) MDGFAGSLDDSISAASTSDVQDRLSALESRVQQQEDEITVLKAALADVLRRLAISEDHVASVKKSVSSKGQPSPRAVIPMSCITNGSGANRKPSHTSAVSIAGKETLSSAAKSGTEKKKEKPQGQREKKEESHSNDQSPQIRASPSPQPSSQPLQIHRQTPESKNATPTKSIKRPSPAEKSHNSWENSDDSRNKLSKIPSTPKLIPKVTKTADKHKDVIINQEGEYIKMFMRGRPITMFIPSDVDNYDDIRTELPPEKLKLEWAYGYRGKDCRANVYLLPTGEIVYFIASVVVLFNYEERTQRHYLGHTDCVKCLAIHPDKIRIATGQIAGVDKDGRPLQPHVRVWDSVTLSTLQIIGLGTFERGVGCLDFSKADSGVHLCVIDDSNEHMLTVWDWQKKAKGAEIKTTNEVVLAVEFHPTDANTIITCGKSHIFFWTWSGNSLTRKQGIFGKYEKPKFVQCLAFLGNGDVLTGDSGGVMLIWSKTTVEPTPGKGPKGVYQISKQIKAHDGSVFTLCQMRNGMLLTGGGKDRKIILWDHDLNPEREIEVPDQYGTIRAVAEGKADQFLVGTSRNFILRGTFNDGFQIEVQGHTDELWGLATHPFKDLLLTCAQDRQVCLWNSMEHRLEWTRLVDEPGHCADFHPSGTVVAI GTHSGRPCCS(SEQ ID NO: 30) MDGFAGSLDDSISAASTSDVQDRLSALESRVQQQEDEITVLKAALADVLRRLAISEDHVASVKKSVSSKGQPSPRAVIPMSCITNGSGANRKPSHTSAVSIAGKETLSSAAKSGTEKKKEKPQGQREKKEESHSNDQSPQIRASPSPQPSSQPLQIHRQTPESKNATPTKSIKRPSPAEKSHNSWENSDDSRNKLSKIPSTPKLIPKVTKTADKHKDVIINQEGEYIKMFMRGRPITMFIPSDVDNYDDIRTELPPEKLKLEWAYGYRGKDCRANVYLLPTGEIVYFIASVVVLFNYEERTQRHYLGHTDCVKCLAIHPDKIRIATGQIAGVDKDGRPLQPHVRVWDSVTLSTLQIIGLGTFERGVGCLDFSKADSGVHLCVIDDSNEHMLTVWDWQKKAKGAEIKTTNEVVLAVEFHPTDANTIITCGKSHIFFWTWSGNSLTRKQGIFGKYEKPKFVQCLAFLGNGDVLTGDSGGVMLIWSKTTVEPTPGKGPKGVYQISKQIKAHDGSVFTLCQMRNGMLLTGGGKDRKIILWDHDLNPEREIEVPDQYGTIRAVAEGKADQFLVGTSRNFILRGTFNDGFQIEVQGHTDELWGLATHPFKDLLLTCAQDRQVCLWNSMEHRLEWTRLVDEPGHCADFHPSGTVVAIGTHSGRRQKHEVNFPKIKLIKKCGMLPGHVAADHPPA VYRRKHQELQAMQMELQSPEYKLSKLRTSTIMTDYNPNYCFAGKTSSISDLKEVPRKNITLIRGLGHGAFGEVYEGQVSGMPNDPSPLQVAVKTLPEVCSEQDELDFLMEALIISKFNHQNIVRCIGVSLQSLPRFILLELMAGGDLKSFLRETRPRPSQPSSLAMLDLLHVARDIACGCQYLEENHFIHRDIAARNCLLTCPGPGRVAKIGDFGMARDIYRASYYRKGGCAMLPVKWMPPEAFMEGIFTSKTDTWSFGVLLWEIFSLGYMPYPSKSNQEVLEFVTSGGRMDPPKNCPGPVYRIMTQCWQHQPEDRPNFAIILERIEYCTQDPDVINTALPIEYGPLVEEEEKVPVRPKDPEGVPPLLVSQQAKREEERSPAAPPPLPTTSSGKAAKKPTAAEISVRVPRGPAVEGGHVNMAFSQSNPPSELHKVHGSRNKPTSLWNPTYGSWFTEKPTKKNNPIAKKEPHDRGNLGLEGSCTVPPNVATGRLPGASLLLEPSSLTANMKEVPLFRLRHFPCGNVNYGYQQQGLPLEAATAPGAGHYEDTILKSKNSMNQPGP

To determine whether a functional EML4-ALK fusion protein was present inthe tumor tissue, immunohistochemistry (IHC) of the tissue with ALK1monoclonal antibodies was performed. IHC confirmed significant ALK1staining of the cytoplasm in tumor cells, indicating overexpression ofthe ALK domain of the fusion protein in the malignant cells (data notshown). Furthermore, nine additional specimens (1 more positive and 8negative by RT-PCR) were confirmed by IHC for a total of 10 of the 55specimens. In total, 2 RT-PCR positive cases were also positive by IHCand 8 RT-PCR negative cases were also negative by IHC.

Using this method to screen a relatively small cohort of NSCLC specimens(n=55) we were able to identify three previously described EML4-ALKfusion variants (1, 3a and 3b) as well as an additional two novelvariants involving exon 17 of EML4 (8a and 8b) in 9.1% (5/55) of theNSCLC specimens examined. Notably, EML4 exon 17 to ALK exon 20 fusionswill not yield an in-frame fusion unless they possesses insertions ordeletions to create an in-frame fusion. In fact, one fusion variant thatwe observed (8a) contained an insertion of 30 bp that results in anearly stop codon and would not likely have malignant transformingactivity on its own. This particular case also expressed variant 8b,with a 95 bp insertion, which is most likely responsible for expressionof the ALK domain in this specimen as observed by IHC and thetransformation or malignant phenotype.

An interesting feature of variant 8b was the presence of a 61 bpsequence of non-adjacent EML4 intron 17. This intron sequence is located˜1.2 kb down stream of exon 17 in the normal EML4 transcript. Based onanalysis of normal lung tissue and non-variant 8b containing cells, itis clear that this configuration of intron 17 is not a result of normalalternative splicing but rather alternative splicing that appears as aresult of translocation.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All nucleotide sequencesprovided herein are presented in the 5′ to 3′ direction.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed.

Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification, improvement and variation of the inventionsembodied therein herein disclosed may be resorted to by those skilled inthe art, and that such modifications, improvements and variations areconsidered to be within the scope of this invention. The materials,methods, and examples provided here are representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

Other embodiments are set forth within the following claims.

1-29. (canceled)
 30. A method of detecting the presence of a nucleicacid encoding a EML4-ALK fusion mutation in a sample comprising EML4-ALKnucleic acids, comprising contacting the sample with a labeled nucleicacid probe that hybridizes to the 5′ insertion junction or the 3′insertion junction of (i) an E17;ins30A20 gene fusion between exon 17 ofEML4 and intron 19 of ALK having a breakpoint region comprising SEQ IDNO: 1, or (ii) an E17ins30;ins65A20 gene fusion between exon 17 of EML4and intron 19 of ALK having a breakpoint region comprising SEQ ID NO:2.31. The method of claim 30, wherein the a labeled nucleic acid probethat hybridizes to the 5′ insertion junction of the E17;ins30A20 genefusion or E17ins30;ins65A20 gene fusion comprises SEQ ID NO:26.
 32. Themethod of claim 30, wherein the a labeled nucleic acid probe thathybridizes to the 3′ insertion junction of an E17;ins30A20 gene fusioncomprises SEQ ID NO:27.
 33. The method of claim 30, wherein the alabeled nucleic acid probe that hybridizes to the 3′ insertion junctionof an E17ins30;ins65A20 gene fusion comprises SEQ ID NO:28.
 34. Themethod of claim 30, wherein the sample is selected from the groupconsisting of: plasma, serum, and biopsy tissue.
 35. The method of claim30, wherein the sample is a lung biopsy sample.
 36. The method of claim30, wherein method comprises nucleic acid amplification.
 37. The methodof claim 30, wherein method comprises real time PCR (RT-PCR).
 38. Themethod of claim 30, wherein method comprises reverse transcriptase PCR.39. The method of claim 30, wherein the sample comprises mRNA.
 40. Themethod of claim 30, wherein the sample comprises cDNA.
 41. The method ofclaim 30, further comprising performing a nucleic acid detection assayon the nucleic acid sample to detect one or more additional EML4-ALKgene fusions selected from the group consisting of: variant 1, variant2, variant 3a, variant 3b, variant 4, variant 5a, variant 5b, variant 6,and variant 7.